It has been known that eukaryotic genomes contain repetitive sequences consisting of similar nucleotide sequence repeats. The first discovered repetitive sequence had a repeat unit consisting of satellite DNA, which is a long sequence ranging from several hundred to several thousand bases, and is called a satellite sequence (Bioscience, 27: 790-796, 1977). Repetitive sequences consisting of shorter nucleotide sequence units were later identified. They have been named, depending on the size of their repeat unit, microsatellite sequences (Am. J. Hum. Genet., 4: 397-401, 1989) when they have a repeat unit of 2 to 5 bases (Nucleic Acids Res. 9: 5931-5947, 1981) or minisatellite sequences (Nature, 314: 67-73, 1985) when they have a repeat unit of 10 to 64 bases (Nature, 295: 31-35, 1982). Microsatellite sequences are also called simple sequences (Nucleic Acids Res. 17: 6463-6471, 1989) or short tandem repeats (Am. J. Hum. Genet. 49: 746-756), etc.
Use of microsatellite sequences as markers was not reported when they were discovered. However, after their polymorphism was confirmed by polymerase chain reaction (PCR) (Am. J. Hum. Genet. 4: 397-401, 1989), they have attracted the attention of researchers for their use as markers in a variety of areas. Specifically, their application to pedigree or lineage discrimination and individual identification of humans, animals, and plants is known. Because microsatellite sequences are scattered throughout a genome and abound in variation, they are good genetic markers. Furthermore, a microsatellite DNA polymorphism contains many polymorphic gene loci and a large number of alleles per gene locus. In addition, based on PCR, microsatellite DNA polymorphism analysis is easy to perform. The amplified products are easily detected as single or double bands by electrophoresis, which simplifies determination of individual types and facilitates the data processing. Therefore, microsatellite DNA markers have become widely used as the most effective markers for population genetics (J. Fish. Biol., 47: 29-55, 1995).
For microsatellite DNA polymorphism analysis, first, a variety of microsatellite DNA should be isolated from individual species to be analyzed. In addition, the microsatellite DNA regions should be amplified by PCR to detect the microsatellite polymorphism. Appropriate primers are needed for PCR. This means that, to perform microsatellite DNA polymorphism analysis, efficient methods for isolating microsatellite DNA from the species and for designing PCR primers capable of amplifying the microsatellite region are required.
The inventors have already reported a method that is expected to allow efficient isolation of microsatellites from poultry, for which microsatellite polymorphism analysis is not advanced (Jpn. Poult. Sci., 33: 292-299, 1996). This method is an improvement on a conventional method of microsatellite sequence isolation that consists of a series of manipulations: fragmentation of a genome with restriction enzymes, insertion of the fragments into a vector, extension reaction with (TG)n primers, and then cloning (Proc. Natl. Acad. Sci. USA 89: 3419-3423, 1992). Namely, isolation of unknown microsatellite sequences was achieved by choosing a vector with high transformation efficiencies, by specifically digesting single-stranded DNA with mung bean nuclease, or by removing bacterial DNA and RNA with DNase I and RNase A. In chicken, it is considered difficult to efficiently isolate microsatellite sequences, because the number of microsatellite sequences is small. The above method could achieve, by calculation, six times more efficient isolation than the known method that was used in an attempt to isolate chicken microsatellite sequences (Poultry Science, 74: 1855-1874, 1995).
However, even by this method, it was impossible to prevent problematic clones from contaminating the microsatellite DNA clones obtained. Namely, a high rate of duplicate clones was found among the clones obtained, suggesting bias in the constitution of clones as well as low efficiency.
Microsatellite sequences are required to be isolated according to each species. There are few species for which the isolation of microsatellite sequences is in progress, and it is still necessary to isolate microsatellile sequences from many species. However, there are a number of species-specific problems, for example, low frequency of microsatellite sequences in the chicken genome. Therefore, it is useful to provide a novel technique for isolating microsatellite sequences, also because it will enable selection from a variety of approaches.